PRIMARY CULTURE OF HUMAN THYROCYTES IN TRANSWELL BICAMERAL CHAMBER - THYROTROPIN PROMOTES POLARIZATION AND EPITHELIAL BARRIER FUNCTION

Epithelial properties of thyrocytes are difficult to maintain in conve ntional cell culture systems. We used bicameral chambers (Transwell(TM )) in attempts to establish a functional epithelium of thyrocytes of h uman origin. Thyroid follicle segments were isolated by collagenase di gestion of paradenomatous tissue obtained at surgery for follicular ad enoma and of tissue from glands with Graves' disease. After careful se paration from connective tissue and single cells by centrifugation, th e follicles were plated at high density on the collagen-coated filter of the chambers and cultured in Eagle's essential medium (EMEM) contai ning 10% fetal calf serum (FCS) or Coon's modified Hams medium enriche d with five or six factors (5H, 6H); the latter media contained 5% FCS without (5H) or with (6H) thyrotropin (TSH). The follicles were conve rted into a confluent cell layer, which had similar DNA content irresp ective of type of medium, after 4-6 days. Cells grown in EMEM or 5H es tablished a transepithelial electrical resistance (R) of 200-500 Omega . cm(2) and was impermeable to [H-3]inulin, indicating the formation of epithelial junctions. Addition of 6H to confluent cells initially c ultured in EMEM or 5H caused a further increase of R, maximally to 150 0 Omega . cm(2), along with a rise of the transepithelial potential di fference; 6H promoted the monolayer formation of cells, increased the number of apical microvilli and reinforced the junctional distribution of actin, cadherin and ZO-1; 6H also enhanced the polarized secretion of [H-3]leucine-labeled thyroglobulin into the apical medium. Cells f rom Graves' thyroid tissue established an epithelium on the filter wit h similar characteristics to that of normal thyrocytes; some platings contained in addition large numbers of HLA-DR positive cells with a de ndritic shape. HLA-DR expression was generally absent in EMEM- or 5H-g rown thyrocytes, but appeared in limited areas of the cell layer after 6H and was expressed by all epithelial cells after interferon-gamma s timulation for 48 h. We conclude that human thyrocytes form a tight an d polarized epithelium when cultured on permeable filters. The polariz ed structure and function of the cells are positively regulated by TSH . The culture system may be useful in studies addressing the role of t he epithelial phenotype (cell polarity and tight barrier) in normal th yroid function as well as in pathological processes in the thyroid, su ch as autoimmunity, cell transformation and tumor progression.